survival and morphology of isolated pancreatic acinar cells from rats with induced acute...

International.Journal ofPancreatolog3', vol. 18, no. 2. 145-152, Oclober 1995 "Q, Copyright 1995 by Humana Press Inc. All rights oFany nature whatsoever reserved. 0169-4197/95/I 8:145 -.152/$5.60

Survival and Morphology of Isolated Pancreatic Acinar Cells from Rats with Induced Acute Pancreatitis Are Not Improved

with Anti-Inflammatory Drugs

F r a n k R & h t e r *'~ a n d R a i n e r Mat th ia s 2

~ Department of Surgeo,, UMD~New Jersey Medical School, Newark, N J." and "Department of Experimental Surgeo', Universi(v '" Otto-von-Guericke. "Magdeburg, Germany

Summary

The influence from anti-inflammatory drugs on cellular damage of pancreatic acinar cells after induction of an acute pancreatitis (AP) in a rat model was investigated. Necrotizing pancreatitis was induced by retrograde instillation of trypsin solution in the pancreatic duct (group I). The severity of inflammation was determined using morphological and histological parameters 6, 24, and 48 h after induction of the necrotizing pancreatitis. After isolation ofacinar cells, the degree of damage was measured by trypan blue exclusion--a parameter of membrane permeability--as well as accumulation ofrhodamine 6G--a parameter of the mitochondrial membrane potential. In groups II-V, rats were treated with the anti-inflammatory drugs indomethacine, hydrocor- tisone, cimetidine, and acetylsalicylic acid (ASS) before induction of AP. There was no significant benefit from therapy in either group regarding cell membrane damage, cellular energy metabolism, or histology.

Key Words: Anti-inflammatory drugs; leukocytes; pancreatic acinar cells; energy state.

Introduction

Acute pancreatitis is still a disease with unknown pathophysiology for which therapy is unsatisfactory. The "primary step" in enzyme activation is still a cause of speculation. Recently, it has been proposed that enzyme-induced pancreatic damage results from excessive stimulation of leukocytes (2).

It is hypothesized that the release of activated hydrolases from pancreatic acinar cells as well as

Received May 9, 1994; Revised February 28, 1995 and April 1, 1995; Accepted April 17, 1995.

*Author to whom all correspondence and reprint requests should be addressed: Department of Surgery, U M D~New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103.

reactive oxygen metabolites is induced by mediators from white blood cells (1,2). The following damage of the pancreas and the severe clinical consequences, like multiorgan failure (MOF), seem to be the result of a vicious cycle that is initiated by prolonged leukocyte stimulation (1,2). To investigate the possible role of leukocyte stimulation, we used sev- eral anti-inflammatory drugs, including hydrocorti- sone, cyelooxigenase inhibitors (acetylsalicylic acid [ASS]), antihistamines (cimetidine), and indomethacine, in an attempt to moderate the effects of invading leukocytes in the pancreatic tissue. The influence of these substances on histological parameters and on energy metabolism of acinar cells isolated from inflamed pancreas was determined.

145

146 Richter and Matthias

Materials and Methods

Animals

Female albino Wistar laboratory rats ( 150-200 g) were used in the experiments. Animals were starved for 24 h before operation. They were anesthetized by ip injection of hcxobarbital (125 mg/kg body wt).

Induction o f AP

AP was induced by intraductal instillation of 0.1 5 mL of trypsin solution (2 mg/mL = 200U in physi- ologic saline) with a microliter syringe over a 3-rain period during ductal clamping (group 1--20 animals). Controls were instilled with 0.1 5 mL of saline.

Groups Ii-IV were treated with anti-inflammatory drugs, single dose 1 h before induction of AP, as follows:

Group 11: Prednisone (3 mg/kg body wt iv) Group llI: ASS (10 mg/kg body wt iv) Group IV: Cimctidine (3 mg/kg body wt iv) Group V: Indomethacine (1 mg/kg body wt iv)

After 6, 24, and 48 h, animals of each group were sacrificed and relaparotomized according to the following protocol:

Time after induction of AP 6 h 24 h 48 h Rats for cell culture/group

I-V (n = 20) 7 7 6 Rats for histology/group

I-V (n = 18) 6 6 6 Macroscopical assessment

of rats/group 13 13 12

The prepared pancreata were used either for cell culture or histoiogic assessment by randomization.

Macroscopic Assessment

The amount of fat necrosis in the retroperitoneum, omentum, and mesentery was graded semiquan- titatively by a scoring system described previously (3). The scores of macroscopic findings were added according to Table 1. The mean sum score was determined for each group. All rats underwent macroscopic assessment.

Histologic Assessment

For histologic assessment, the pancreata of the animals were removed, fixed in 10% formalin, and embedded in paraffin. Because of the nonuniform histologic changes throughout the pancreas, four

Table 1 Macroscopic Scoring

(from Spormann and Sokolowski [3])

Findings Evaluation Score

Fat necroses Light 1 (lesser sac/omentum) Medium 2

Severe 3 Fat necroses Light 1

(peripancreatic) Medium 2 Severe 3

Fat necroses Light 1 (retroperitoneal) Medium 2

Severe 3 Parenchymal Focal 1

necroses Sublobular 2 Lobular 3

Hemorrhages Light 1 Medium 2 Severe 3

Table 2 Histologic Scoring

(from Spormann and Sokolowski [3])

Findings Evaluation Score

Edema Light 1 Medium 2 Severe 3

Inflammatory Light 1 infiltrate Medium 2

Severe 3 Fat necroses <2 3

3-5 5 >5 7

Parenchymal Focal 3 necroses Sublobular 5

Lobular 7 Hemorrhages Light 3

Medium 5 Severe 7

horizontal step sections were taken from each block and stained with hematoxylin and eosin. The histologic findings were evaluated by a quantitative scoring system (Table 2) as described previously (3). For each group, the mean sum score was determined.

Separation o f Pancreatic Acinar Cells

Pancreatic acinar cells were isolated using collagenase digestion and disruption ofdesmosomes with

International Journal of Pancreatology Volume 18, 1995

AP and Anti-Inflammatory Drugs 147

Ill

!D"

Q

]'

Fig. 1. Electronmicroscopic photographs of pancreatic acinar cells from rat pancreas (A) during isolation procedure after primary collagenase digestion (above left), (B) isolated acinar cell in culture with zymogen granula at the apex and microvilli (above right), (C) still intact isolated acinar cells after 4 h of incubation in Eagles' medium (below left), and (D) damaged acinar cells in culture medium with "blebs" (below right).

EDTA and separation of the acinar cells by density gradient (4). Cells were incubated in Eagles' medium with 10 mM HEPES in a 50-mL Erlenmeyer flask, gassed with oxygen, and agitated at 37~ in a shak- ing water bath.

At specified time intervals, aliquots were withdrawn, and the number of intact cells and those stained with trypan blue were counted in a Neubauer chamber. The integrity of acinar cells was evaluated morpho- logically after fixation of aliquots in glutaraldehyde by electron microscopy with special attention given to cell polarization and presentation ofmicroviili (Fig. 1).

Assessment o f Cellular Energy State Changes of the mitochondrial membrane poten-

tial were monitored by measurement of rhodamine 6G fluorescence. Rhodamine 6G as a cationic fluorophore is accumulated selectively by mitochondria of living cells in proportion to the mitochondrial membrane potential (5). At specified times, triplicate samples of the cell cultures were withdrawn and mixed with rhodamine 6G at a final concentration of 3.3 mg/L. Since some portion of the fluorophore is bound nonspecifically at the cell membrane, samples of cells were uncoupled with excess carbonyl-

International Journal of Pancreatology Volume 18. 1995


6 24 time in (hours)

48

Fig. 2. Macroscopic mean sum scores after trypsin- induced pancreatitis. Each column represents the mean of 13 rats (6-24 h) and 12 rats (48 h). [ ] Group l--untreated, �9 group ll---cortisone,17/A group III--ASS, []group IV-- cimetidine, and [::qgroup V--indomethacine.

,i

6 24 48 time in (hours)

Fig. 3. Histologic mean sum scores after trypsin- induced pancreatitis. Each column represents the mean of six rats. 7--1 Group l--untreated, I group lI---cortisone, [ ] group III--ASS, m group IV---cimetidine, and [::q group V--indomethacine.

cyan ide-m-chlorophenylhydrazone (CCCP) and used as blanks. After 15 min of incubation and sub- sequent centrifugation (1000g) the accumulation of rhodamine 6G by acinar cells was determined fluo- rimetrically (318/520-nm wavelengths for emission and transmission).

Results

Morphologic Scor ing

At 6 h after instillation of trypsin, there were no macroscopic changes in any group. At 24 and 48 h, all animals showed typical signs of pancreatitis as extrapancreatic fat neuroses, hemorrhages, and ascites. Figure 2 represents the macroscopic sum scores in each group. There was no statistically significant difference between the groups (Wilcoxon test,p < 0.01 ). Histologically at 6 h after trypsin instillation, only perilobular edema and beginning infiltration of ieu- kocytes (group I only) were detectable. At 24 and 48 h, parenchymal necroses, inflammatory infiltration, and hemorrhages were regularly found in each group (Fig. 3). The histologic changes were found to be focal and not uniform throughout the pancreas. The degree of leukocyte inflammation was lowest in the group treated with cortisone (counted leukocytes/vision field). However, the difference ofleukocytes/vision field among the groups was not statistically significant.

100

80

60

40

20

Y'~ld (MIo cells per pancreas)

6 24 48

time in (hours)

Fig. 4. Yield of isolated pancreatic acinar cells from rats with trypsin-induced pancreatitis. Each column represents the mean of 7 rats (6-24 h) and 6 rats (48 h) with different treatment modalities (groups 1-V).l--IGroup l - - untreated, i group ll---cortisone, [ ] group II1-ASS, �9 group IV--cimetidinc, andF:qgroup V--indomethacine.

Cell Isolation and Yield

The yield of isolated acinar cells in relation to treatment and time after induction ofpancreatitis is shown in Fig. 4. The number o f cells harvested from untreated animals with comparable body weight is 100-120 x 106. At 6 h after induction ofAP, a reduc-

International Journal of Pancreatology Volume 18. 1995


Intact cells (%)

. I 100 ~P---'--'~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I

80

60

40

20

0

+ , [

60 120 180 240

time (min)

Fig. 5A. Viability of acinar cells during 4 h of incubation in Eagle's medium in dependence on time after induction of AP. Each point represents the mean of 7 (6-24 h) or 6 (48 h) measurements with trypan blue counting. Group I--untreated. --~ 6 h , n 24 h, and-*-48 h.

~tact cells (%) 120

B 100,

i 1, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I .I, m 1,

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

+4o+ �9 ...... ' - - , 0

0 60 120 180

time (min)

240

Fig. 5B. Viability ofacinar cells during 4 h of incubation in Eagle's medium in dependence on time after induction of AP. Each point represents the mean of 7 (6-24 h) and 6 (48 h) measurements with trypan blue counting. Group II-- cortisone.--k--6 h, 41-24 h, and-*-48 h.

tion of cell yield is obvious despite only minimal morphologic changes in the pancreas. At 24 and 48 h after induction of AP, the yield of harvested cells is reduced to one-third of the amount in untreated animals. There is no statistically significant difference between the groups treated with anti-inflammatory drugs (Wilcoxon test, p < 0.01).

Cell Survival in Culture

The survival of cells in culture medium during a 4-h period, 6 h after induction of AP, was not different from that of untreated animals. At 24 and 48 h, the cell viability was minimal in all groups (0% survival

within 4 h of incubation in group I and 5-15% survival in groups II-V). The difference in survival between group I (no treatment) and groups I I -V (treatment before induction of AP) is not statistically significant (Wilcoxon test p < 0.05, n = 8/group) (Fig. 5A-E). Control suspensions ofacinar cells from untreated rats had an initial viability of 90-95% and lost <10% of their initial viability during 4 h of incubation.

Cellular Mitochondrial Membrane Potential

The mitochondrial membrane potential of isolated acinar cells measured by rhodamine 6G accumula-



Intact cells (%) : ~ o ~ _ ~ ...... , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c

t . . . . . . . L _ , i ............. ' . . . . . . ' ' ......... '

0 60 120 180 240

time (min)

Fig. 5C. Viability of acinar cells during 4 h incubation in Eagle's medium in dependence on time after induction of AP. Each point represents the mean of 7 (6-24 h) and 6 (48 h) measurements with trypan blue counting. Group III--ASS. --1r 6 h, I 2 4 h, and -~-48 h.E

Intact cells (%) 120

D

1 0 0 ' . . . . ~ , ~ ~ ,

80 =

oo .................................... l i , ~ 1 ............. , . . . . . . . . . . . . 40 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

0 0 60 120 180 240

time (min)

Fig. 5D. Viability of acinar cells during 4 h of incubation in Eagle's medium in dependence on time after induction of AP. Each point represents the mean of 7 (6-24 h) and 6 (48 h) measurements with trypan blue counting. Group IV-- cimetidine.--~-- 6 h, t - 2 4 h, and-~48 h.

Intact cells (%) 120

100' I I o i j, , ,L I

8 o . . . . . . . !~ . . . . = ~ = ' ~ = ~ i . . . . . . . . -

I 60

E

40

20 ........... . . . . . . �9 .... i ii

0 . . . . . . . .

0 60 120 180 240

t i m e ( r a i n )

Fig. 5E. Viability of acinar cells during 4 h of incubation in Eagle's medium in dependence on time after induction of AP. Each point represents the mean of 7 (6-24 h) and 6 (48 h) measurements with trypan blue counting. Group V-- indomethacine. --#- 6 h, I 2 4 h, and -~-48 h.



dTmax (mm/Mio cells) 140

1201 O0804060

2O 0 6 24 48

time in (hours)

Fig. 6. Maximum rhodamine 6G accumulation by acinar cells isolated at various times after trypsin-induced pancreatitis. Each column represents the mean of 7 rats (6-24 h) and 6 rats (48 h) at different treatment modalities. [ ] Group I--untreated, [ ] group II---cortisone, [ ] group III--ASS, [ ] group IV---cimetidine, and [ ] group V-- indonaethacine.

tion in dependence on treatment is shown in Fig. 6. The maximum rhodamine accumulation over 4 h of incubation was reduced in cells harvested from animals at 6 h after induction of AP despite only minimal morphologic changes. The minimum of rhodamine accumulation was reached at 24 and 48 h after induction of AP. There was no significant difference between treated (groups II-V) and non- treated animals (group t) (Wilcoxon test, p < 0.01, n = 8/group).

Discussion

The time-course of morphologic and enzymatic changes within 6 d after retrograde trypsin instillation in the pancreatic duct of the rat has recently been described by Letko et al. (5). It was shown that histologic changes of the pancreas correlated well with cell survivability and the mitochondrial potential of isolated cells in culture after induction of AP. It was also shown that when the membrane permeability of isolated cells was minimal, at 24-48 h after trypsin instillation, the migration ofleukocytes into the pancreas reached the highest level (5).

These findings support the postulated role of leukocytes in the induction of necrotizing pancreatitis

as described by Rinderknecht (2) and Banks et al. (1). These authors hypothesized that the complex pro- cess of induction of severe necrotizing pancreatitis primarily results from excessive stimulation of leukocytes. There is some evidence regarding this assumption because mediators from white blood cells, such as TNFGt, I1-6, I1-8, and neutrophile elastase, are elevated in acute necrotizing pancreatitis in the human (3, 6-9).

If leukocyte activation is assumed to be an induc- ing factor in the pathogenesis of acute pancreatitis, it should be possible to moderate the effects of mediator release with anti-inflammatory drugs (10). How- ever, in our model of trypsin-induced pancreatitis in the rat, we were not able to find a benefit from anti- inflammatory agents either in histologic changes or in parameters of cellular physiology. The amount of immigrating neutrophils in the cortisone-treated group was lower than in the other groups (counted round cells/vision field), but the histologic sum score was not significantly different.

Furthermore, a slight survival benefit seemed to occur after isolation and incubation of acinar cells from animals treated with cortisone. However, the difference in 4-h survival in Eagle's medium of 0 vs 5-15% was small and not statistically significant.

No difference between the groups was seen regarding the mitochondrial membrane potential. However, results should be interpreted cautiously because the doses of the anti-inflammatory drugs were adopted from the usual doses in humans and are not necessarily the optimal doses for the rat. The medi- cations were given in a single dose to ensure equal conditions in each group. Long-term treatment in this in vitro model ofnecrotizing pancreatitis, in analogy to the usual clinical practice, does not seem to be helpful, because it has been shown earlier that the morphologic characteristics of the induced pancreatitis change after 4-6 d toward a "chronicity" of the histologic lesions (5).

It is still unclear which of the many mediators that are released from neutrophils are responsible for cellular damage and ifneutrophils alone are responsible for the initiation of the pancreatic enzyme digestion. The experiments of Schulz and Niederau with pancreatic acinar cells coincubated with activated neutrophils failed to detect any damaging effect of these leukocytes alone (11). Further investigations at the



cellular level with more specifically acting mediator blocking substances will be needed to elucidate the role of leukocytes in acute pancreatitis.

References

1 Banks RE, Evans SW, Alexander D, Mc Mahon M J, Whicher JT. Is fatal pancreatitis a consequence of excessive leukocyte stimulation? The role of tumor necrosis factor alpha. Cytokines 1991; 3: 12-16.

2 Rinderknecht H. Fatal pancreatitis, a consequence of excessive leukocyte stimulation? IntJPancreato11988; 3: 33-44.

3 Sporrnann H, Sokolowski A, Letko G. Effect of temporary ischemia upon development and histological patterns of acute pancreatitis in the rat. Pathol Res Pract 1989; 184: 507-513.

4 Schulz HU, Letko G, Spormann H, Sokolowski A, Kemnitz P. An optimized procedure for isolation of rat pancreatic acinar cells. Anat Anz 1988; 167: 141-150.

5 Letko G, Richter F, Matthias R. Influence of trypsin- induced acute pancreatitis on survival and energy state of isolated acinar cells from rat pancreas. Pathol Res Pract 1992; 188: 205-210.

6 Chung KC, Barnes PJ. Platelet activating factor: A potent mediator of inflammation. PostgradMed 1989; 65: 420,421.

7 Fujimura K, Kubota Y, Ogura M, Yamaguchi T, Binnaka T, Tani K, Kitagawa S, Mizuno T, Inoue K. Role of endoge- nous platelet-activating factor in caerulein-induced acute pancreatitis in rats: protective effects ofa PAF-antagonist. J Gastroenterol Hepatol 1992; 7: 199-202.

8 Gross A, Andreesen R, Leser HG, Ceska M, Liehl E, Lausen M, Farthmann EH, Scholmerich J. Interleukin-8 and neutrophil activation in acute pancreatitis. EurJClin Invest 1992; 22: 200-203.

9 Heath DI, Cruickshank A, Gudgeon M, Jehanli A, Shenkin A, Imrie CW. Role ofinterleukin-6 in mediating the acute phase protein response and potential as an early means of severity assessment in acute pancreatitis. Gut 1993; 34: 41-45.

10 Van Ooijen B, Ouwendijk RJ, Kort W J, Zijlstra F J, Vincent JE, Wilson JH, Westbroek DL. Raised plasma thromboxane B2 levels in experimental pancreatitis in rats. The effects of flunarizine, dazoxiben and indomethacine. Scand J Gastroenterol 1988; 23:188-192.

11 Schulz HU, Niederau C, Hinze D. Mechanisms of ethanol- induced pancreatic injury: Insights from in vitro studies using isolated rat pancreatic acinar cells. Abstract 117. XXVth Congress of the European Pancreatic Club, Paris 1993.


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